The tuatara (Sphenodon punctatus)-the only living member of the reptilian order Rhynchocephalia (Sphenodontia), once widespread across Gondwana 1,2-is an iconic species that is endemic to New Zealand 2,3. A key link to the now-extinct stem reptiles (from which dinosaurs, modern reptiles, birds and mammals evolved), the tuatara provides key insights into the ancestral amniotes 2,4. Here we analyse the genome of the tuatara, which-at approximately 5 Gb-is among the largest of the vertebrate genomes yet assembled. Our analyses of this genome, along with comparisons with other vertebrate genomes, reinforce the uniqueness of the tuatara. Phylogenetic analyses indicate that the tuatara lineage diverged from that of snakes and lizards around 250 million years ago. This lineage also shows moderate rates of molecular evolution, with instances of punctuated evolution. Our genome sequence analysis identifies expansions of proteins, non-protein-coding RNA families and repeat elements, the latter of which show an amalgam of reptilian and mammalian features. The sequencing of the tuatara genome provides a valuable resource for deep comparative analyses of tetrapods, as well as for tuatara biology and conservation. Our study also provides important insights into both the technical challenges and the cultural obligations that are associated with genome sequencing.

BackgroundContiguous genome assemblies are a highly valued biological resource because of the higher number of completely annotated genes and genomic elements that are usable compared to fragmented draft genomes. Nonetheless, contiguity is difficult to obtain if only low coverage data and/or only distantly related reference genome assemblies are available.FindingsIn order to improve genome contiguity, we have developed Cross-Species Scaffolding—a new pipeline that imports long-range distance information directly into the de novo assembly process by constructing mate-pair libraries in silico.ConclusionsWe show how genome assembly metrics and gene prediction dramatically improve with our pipeline by assembling two primate genomes solely based on ∼30x coverage of shotgun sequencing data.

Diatoms are among the most commonly used bioindicators. Correct taxonomic identifications are critical to their use as bioindicators because closely related diatom species can respond differently to water physicochemical characteristics and pollutants. However, diatom identification based on morphology can be time consuming, and requires highly specialized taxonomic skills. To optimize diatom identification, DNA metabarcoding is increasingly used because it is generally less time consuming and may be more accurate than morphological identification. To date, however, neither DNA metabarcoding nor DNA barcoding diatom studies have been conducted in Mexico. Thus, we studied epilithic diatoms from streams in Central Mexico with a combination of morphological and metabarcoding techniques, and compared the diatoms identified and quantified by each method. We also assembled a barcode reference library based on clonal culturing. This library is composed of 190 strains that belong to 72 species in 24 genera. The morphological analysis of environmental samples resulted in the identification of 204 infrageneric taxa in 42 genera, but clonal culturing from the same samples retrieved 12 additional infrageneric taxa and 1 additional genus, thereby revealing concealed diversity. The metabarcoding approach resulted in the identification of 266 infrageneric taxa that belonged to 35 genera. Together, these methods detected 49 genera. Of these genera, 14 were identified only by morphology, 29 were identified by both methods, and 6 were only identified by metabarcoding. Of the 266 taxa we retrieved with metabarcoding, we confidently assigned 94 infrageneric taxa because a direct morphological or barcode sequence correlation was possible. Thirty-four of these 94 taxa were only detected with the metabarcoding method. One-fourth (23) of the assignments were only possible because of the barcode reference library we developed during this study, because there were no existing barcode sequences that matched these barcodes in the International Nucleotide Sequence Database Collaboration databases. Large disparities existed between relative abundances based on valve counts and sequence reads of the most abundant taxa, even after we corrected for cell biovolume. Overall, we conclude that the combination of morphological and molecular methods increases the detection and identification of diatoms.